Phosphorylation of protein kinase C (PKC) substrates in T lymphocytes was analyzed after stimulation with specific pairs of anti-CD2 monoclonal antibodies (mAb) or an anti-CD3 mAb. The results show that the appropriate stimulation of both CD2 or CD3 antigens results in phosphorylation of a 80-kDa putative PKC substrate and that this phosphorylation event is sensitive to a PKC inhibitor, sphinganine. CD2- and CD3-dependent phosphorylation was found to be strongly dependent on an extensive cross-linking of surface antigens. The biological importance of cross-linking of CD2 and CD3 was also evident for other biological responses such as interleukin 2 production and induction of an autocrine growth response. Finally, we also present evidence for interaction between the CD2 and CD3 signal transducing pathways.